The Na+-Ca2+ exchanger of cardiac sarcolemma is the dominant Ca2+ efflux mechanism in the myocardium. As such, the Na+-Ca2+ exchanger has a critical role in the regulation of cardiac contractility. Further molecular knowledge of this essential cardiac transporter will help understand both normal and diseased hearts. The long-term goal of this project is to understand the molecular mechanisms involved in the transport process. Towards this goal, the specific aims of the project are as follows: 1. Mutagenesis. This laboratory has recently cloned the cardiac Na+-Ca2+ exchanger. The importance of specific amino acids will now be analyzed by site-directed mutagenesis. Mutants will be expressed in oocytes and analyzed by the giant excised patch technique. 2. Topology. Immunological approaches will be used to determine the arrangement of the Na+-Ca2+ exchange protein in the sarcolemmal membrane. 3. Mapping of binding sites. The Na+-Ca2+ exchanger is capable of binding calmodulin, exchanger inhibitory peptide, regulatory Ca2+, and ankyrin with high affinity. The amino acids involved in the binding of these ligands to the exchanger will be determined by a unique sublibrary approach. 4. Interaction with cytoskeleton. the Na+-Ca2+ exchanger is constrained to specific domains of the sarcolemma by interactions with the cytoskeleton. These interactions of the exchanger with components of the cytoskeleton will be characterized. 5. Protein biochemical approaches. Recombinant exchanger protein will be purified for initial structural studies of the exchanger protein.